Reaction of (di)amines in the presence of a lysine oxidase and of a reducing agent

ABSTRACT

The invention relates to methods for reacting (di)amines as substrates in the presence of a lysine oxidase arid a reducing agent, resulting in alcohols, diols or cyclic secondary amines. In a particular embodiment, the invention is directed to methods of preparing cyclic secondary amines suitable for ultimately synthesizing piperidine-2-carboxylic acid and proline derivatives, useful, for example as thrombin inhibitors.

RELATED APPLICATIONS

This application claims the benefit of German Application Ser. No.10117730.5, filed Apr. 9, 2001, the entire contents of which areincorporated herein by this reference.

The invention relates to methods for reacting (di)amines as substratesin the presence of a lysine oxidase and of a reducing agent. Dependingon the substrate, this results in alcohols, diols or cyclic secondaryamines.

The invention further relates to the use of a lysine oxidase in saidmethods.

Proline derivatives are important intermediates for thrombin inhibitors(WO 95/35309, WO 98/06740, WO 94/29336), and piperazine-2-carboxylicacid derivatives are used in HIV protease inhibitors. Preparation ofsuch cyclic secondary araines such as piperazine-2-carboxylic acid,piperidine-2-carboxylic acid and proline derivatives frequently requiresgreat synthetic complexity, as described by D. Askin et al. inTetrahedron Lett. 1994, 35(5), 673–676 and in WO 98/04523. A method forpreparing partly unsaturated piperidine-2-carboxylic acid derivativeswhich is rather simple by comparison starts from L-lysine. Its α-aminogroup is oxidized in the presence of oxygen to the keto group withformation of H₂O₂ and NH₃, to result in 2-oxo-6-aminohexanoic, asdescribed in J. Basic Microbiol. 1994, 34(4), 265–276. If this reactiontakes place in the presence of catalase, the resulting2-oxo-6-aminohexanoic acid is cyclized to1,2-didehydropiperidine-2-carboxylic acid. In order to preparepiperidine-2-carboxylic acid therefrom it would be necessary to reducethe 1,2-didehydropiperidine-2-carboxylic acid in an additional processstep after purification.

It is an object of the present invention to find a further method forpreparing cyclic secondary amines which is suitable for synthesizingpiperidine-2-carboxylic acid and proline derivatives.

We have found that this object is achieved by a method for reactingamines of the general formula (I)

with A- meaning

to give compounds of the general formula (II) or (III)

with B- meaning HO—CH₂—R³— or R⁴—comprising the following steps:

-   -   a) oxidation of at least one

-   -    group of an amine of the general formula (I) to a carbonyl        group in the presence of a lysine oxidase as catalyst;    -   b) where appropriate reaction of the second NH₂ group of an        amine of the general formula (I) with

With the carbonyl group resulting from a) to give an enamine or imine bycyclization;

-   -   c) reduction of the carbonyl group(s) resulting from a), or of        the enamine or imine resulting where appropriate from b), with a        reducing agent respectively to hydroxymethyl groups or to a        cyclic secondary amine of the general formula (II),        where R¹ is NR⁵ or a linear bivalent C₂–C₆ hydrocarbon radical        which is optionally substituted by CO₂H, CO₂R⁶, OH, SH and/or        N(R⁵)₂ and/or contains nonadjacent O, S and/or N atoms,        where R⁵ is H or linear C₁–C₆-alkyl and R⁶ is linear C₁–C₆-alkyl        or branched C₃–C₆, C₆–C₁₀-aryl,        and R² is H, CO₂H, CO₂R⁶ or CN,        where the method is carried out as a one-pot method, lysine        oxidase and reducing agent are present together, and        in the definition of A in the general formula (I)        R³ is a bivalent C₀–C₁ or C₅–C₁₈ hydrocarbon radical which is        optionally substituted by linear C₁–C₆-alkyl or branched        C₃–C₆-alkyl, C₆–C₁₀-aryl, C₄–C₁₀-heteroaryl and/or CO₂R⁶ and/or        contains nonadjacent O, S and/or N atoms, and        R⁴ is a linear C₁–C₂₀ hydrocarbon radical which is optionally        substituted by functional groups such as CO₂H and/or CO₂R⁶        and/or contains nonadjacent O, S and/or N atoms.

It is all the more surprising to carry out the method of the inventionas one-pot method since it was not to be expected that lysine oxidasesretain their activity in the presence of a reducing agent. Purificationof the cyclic enamines or imines resulting from b) or the compounds withat least one carbonyl group resulting from a) before reduction to cyclicsecondary amines of the general formula (II) or alcohols of the generalformula (III) is therefore unnecessary. In addition, step b) of themethod of the invention, the cyclization to the enamine or imine, takesplace spontaneously even without addition of catalase, in contrast tothe known method.

Depending on the structure of the amine of the general formula (I), thesubstrate, the individual steps of the method of the invention proceedin detail as follows:

1) If an amine of the general formula (I) with

where R¹ and R² have the abovementioned meaning is employed, thesubstrate is a diamine. In step a) of the method of the invention, oneof the two

groups of this diamine is oxidized to a carbonyl group with catalysis bya lysine oxidase. It has been observed that one use of the lysineoxidase from Pichia pastoris there is oxidation exclusively of theε-amino group and not of the α-amino group of L- and D-lysine. However,it is immaterial for the following steps which amino group has beenoxidized or whether an aldehyde or a ketone is present. The carbonylgroup generated in step a) then reacts spontaneously in step b) with thesecond—unreacted—amino group to give a Schiff's base (an imine) or anenamine. Since this is an intramolecular reaction it results in a cyclicenamine or imine. The latter can also be isolated. The method of theinvention is, however, such that an isolation is unnecessary before stepc) because steps a) to c) are carried out as a one-pot method. In stepc), the cyclic enamine or imine is then reduced by a reducing agentpresent in the substrate solution to a cyclic secondary amine of thegeneral formula (II).

2) If an amine of the general formula (I) with A-=H₂N—CH₂—R³— where R³has the abovementioned meaning is employed, the substrate is a diaminewith two aminomethyl groups. Both aminomethyl groups are oxidized instep a) of the method of the invention in the presence of a lysineoxidize as catalyst to formyl groups. In contrast to 1), anintramolecular reaction is not possible to not favored and thereforedoes not take place. Both formyl groups are then reduced in step c) witha reducing agent to hydroxymethyl groups to form an alcohol of thegeneral formula (III) with B-=HO—CH₂—R³—.

3) If an amine of the general formula (I) with A=R⁴ where R⁴ has themeaning indicated above is employed, its aminomethyl group is oxidizedto a formyl group. This formyl group is then reduced in step c) of themethod of the invention by the reducing agent present in the substratesolution to a hydroxymethyl group to form a primary alcohol of thegeneral formula (III) with B=R⁴.

The methods of the invention can be described in detail as follows:

1) Method for preparing cyclic secondary amines of the general formula(II) from diamines of the general formula (Ia)

where R¹ is NR⁵ or a linear bivalent hydrocarbon radical with 2 to 6 Catoms, which is optionally substituted by functional groups such asCO₂H, CO₂R⁶, OH, SH and/or N(R⁵)₂, and/or contains nonadjacentheteroatoms such as O, S, N,where R⁵ is H or linear C₁–C₆-alkyl and R⁶ s linear C₁–C₆-alkyl orbranched C₃–C₆-alkyl C₆–C₁₀-aryl, and R² is H, CO₂H, CO₂R⁶ or CN,comprising the following steps:

-   -   a) oxidation of one of the two

-   -    groups of a diamine of the general formula (Ia) to a carbonyl        group in the presence of a lysine oxidase as catalyst;    -   b) reaction of the second NH₂ group of the diamine of the        general formula (Ia) with the carbonyl group resulting from        step a) to give an enamine or imine by cyclization;    -   c) reduction of the enamine or imine resulting from b) with a        reducing agent to give a cyclic secondary amine of the general        formula (II);        wherein the method is carried out as one-pot method, and lysine        oxidase and reducing agent are present together.

In the definition of the radical R¹, the term “hydrocarbon radical” alsoencompasses (partially) unsaturated hydrocarbon radicals with cis doublebonds.

Examples of suitable diamines of the general formula (Ia) are basicamino acids such as D- and L-lysine and D- and L-ornithine,2,6-diamino4-azahexanoic acid, and 1,6-diaminohexane. L-lysine andL-ornithine are preferably employed. Use of L-lysine results inpiperidine-2-carboxylic acid, and use of L-omithine results in proline.

2) Method for preparing diols of the general formula (IIIa) fromdiamines of the general formula (Ib)

comprising the following steps:

-   -   a) oxidation of the two aminomethyl groups of a diamine of the        general formula Ib) to formyl groups in the presence of a lysine        oxidase as catalyst;    -   b) reduction of the formyl groups resulting from a) with a        reducing agent to hydroxymethyl groups to form a diol of the        general formula (IIIa);        wherein the method is carried out as one-pot method, lysine        oxidize and reducing agent are present together, and        R³ is a bivalent C₀–C₁ or C₅–C₁₈ hydrocarbon radical which is        optionally substituted by linear C₁–C₆-alkyl or branched        C₃–C₆-alkyl, C₆–C₁₀-aryl, C₄–C₁₀-heteroaryl and/or CO₂R⁶, and/or        contains nonadjacent O, S and/or N atoms.

The term “bivalent hydrocarbon radical” also includes in this connectionaryl, alkylaryl, (alkyl)cycloalkyl and optionally partially unsaturated(alkyl)heterocycloalkyl groups. The term “heteroaryl” includes partiallyor completely unsaturated cyclic systems which contain 4 to 10 C atomsand one or more nonadjacent O, N or S atoms or N(H) groups.

The compounds of the general formula (Ib) which are preferably employedare those in which R³ is a linear bivalent C₀–C₁ or C₅–C₁₈ hydrocarbonradical which is optionally substituted by CO₂R⁶ and/or containsnonadjacent O, S and/or N atoms.

Examples of suitable diamines of the general formula (Ib) areethylenediamine, 1,4-diaminobutane, 1,8-diaminooctane and spermine.

3) Method for preparing primary alcohols R⁴—CH₂—OH from aminesR⁴—CH₂—NH₂ comprising the following steps:

-   -   a) oxidation of the aminomethyl group of an amine R⁴—CH₂—NH₂ to        a formyl group in the presence of a lysine oxidase as catalyst;    -   b) Reduction of the formyl group resulting from a) with a        reducing agent to a hydroxylmethyl group to form a primary        alcohol R⁴—CH₂—OH;        wherein the method is carried out as one-pot method, lysine        oxidize and reducing agent are present together, and        R⁴ is a linear C₁–C₂₀ hydrocarbon radical which is optionally        substituted by functional groups such as CO₂H and/or CO₂R⁶ (with        R⁶=linear C₁–C₆-alkyl or branched C₃–C₆-alkyl or C₆–C₁₀-aryl)        and/or contain nonadjacent O, S and/or N atoms.

The term “linear hydrocarbon radical” also includes linear (alkyl)arylgroups, linear (alkyl)cycloalkyl groups and linear(alkyl)heterocycloalkyl groups.

Examples of suitable amines R⁴—CH₂—NH₂ are n-butylamine, ethanolamine,glycine ethyl ester, 3-aminomethylpyridine and benzylamine.

The invention also relates to the use of a lysine oxidase in methods forreacting (di)arnines to give cyclic secondary amines, diols or alcoholsin the presence of a reducing agent.

Lysine oxidases catalyze in vivo the oxidation of the ε-amino group oflysine to aldehydes and thus initiate the stabilization of colagen andelastin fibers through formation of covalent crosslinks (Am. J. Respir.Cell Mol. Biol. 1991, 5, 206–210).

The lysine oxidases (E.C. class 1.4.3) generally employed for themethods of the invention are of microbial origin, i.e. from eukaryotessuch as fungi or yeasts or prokaryotes such as Gram-positive orGram-negative bacteria or archaebacteria. Lysine oxidases from yeasts ofthe general Candida, Hansenula, Pichia, Sporobolomyces, Sporopachydermiaor Trigonopsis are preferably used. Particularly preferred lysineoxidases are those from the general and species Candida nagoyaensis,Candida nemodendra, Candida boidinii, Candida lipolytica, Candidasteatolytica, Candida tropicalis, Candida utilis, Hansenula minuta,Hansenula polymorpha, Pichia pinus, Pichia pastoris, Sporobolomycesalborubescens, Sporopachydermia cereana or Trigonopsis variabilis. Thelysine oxidase from the genus and species Pichia pastoris is veryparticularly preferred for the method of the invention.

It is possible to employ both unpurified crude enzymes and purifiedenzymes. All organisms or cells can also be used for the method of theinvention as long as the enzymes are secreted into the extracellularmedium or the cells have been permeabilized. The method of the inventionis preferably carried out with purified enzymes.

It is advantageous that lysine oxidases can be obtained on the largescale by fermentation processes. For example, lysine oxidases fromyeasts are obtained by fermenting the yeast which secretes the desiredlysine oxidase in a nutrient medium containing yeast extract, soybeanoil and conventional additives such as mineral salts and trade elementsand, where appropriate, buffer substances. After the fermentationprocess, the lysine oxidases are released by a cell disruption which iscarried out by conventional methods known to the skilled worker. Aftersubsequent centrifugation, the supernatant solution is purified by ionexchange chromatography with subsequent molecular sieve chromatographyand then a further ion exchange chromatography. It is possible therebyto prepare lysine oxidases with a purity of more than 90%, preferably ofmore than 95%, particularly preferably of more than 99%. In place ofion-exchange chromatography and molecular sieve chromatography it isalso possible to purify by hydrophobic chromatography and precipitationmethods.

For the reaction of the (di)amines in the methods of the invention, thelysine oxidase is generally employed in a concentration of from 2 to 20units per mmol of substrate. 1 unit is defined as the amount of enzymewhich catalyzes the formation of 1 μmol of H₂O₂ per minute at 30° C.

The reducing agents employed are generally those allowing the reactionto be carried out in aqueous medium. Examples thereof are the alkalimetal and alkaline earth metal borohydrides, triacetoxyborohydrides,cyanoborohydrides and dithionites. In place of the alkali metal oralkaline earth metal salts, it is also possible to employ the transitionmetal salts of transition metals such as zinc, iron, manganese. The useof alkali metal salts is preferred, especially of NaBH₄. The reducingagent is generally employed in a concentration of ≦1% by weight,preferably in a concentration of ≦0.5% by weight based on the totalamount of reaction medium. The total amount of reaction medium comprisesthe solution in which the reaction is carried out and, whereappropriate, buffer substances, substrate, lysine oxidase and reducingagent present therein.

The reaction is generally carried out in aqueous solutions containingbuffer substances. Suitable buffer substances are all buffer substanceswhich buffer a pH range from 6.5 to 7.4, preferably a pH range from 6.8to 7.2. Suitable buffer substances are the organic buffer substancesknown as Good's buffers, and phosphate/diphosphate buffers. Buffersubstances containing amino groups, and potassium sodiumphosphate/disphosphate are preferred, and trishydroxymethylaminomethane(called TRIS buffer) is particularly preferred. Other suitable buffersubstances which buffer in the range between pH 6.5 and pH 7.4 can befound in standard works of reference.

In one embodiment of the invention, the reaction is carried out byadding the (di)amine to the stirred buffer solution containing lysineoxidase and reducing agent. In another embodiment of the invention, thelysine oxidase and the reducing agent are added simultaneously to thestirred (di)amine.

Said reducing agents are for the most part commercially available assolutions or suspensions in organic solvents such as glymes (glycolethers), lower alcohols or dioxane, and are employed in this form.However, it is also possible to employ aqueous solutions of the reducingagents.

Addition of the reactants can take place both continuously anddiscontinuously, and preferably takes place discontinuously.

The lysine oxidase can be recovered for example by ultrafiltration andchromatography.

The method of the invention can generally be carried out at temperaturesbetween 0 and 60° C., preferably between 10 and 40° C., particularlypreferably between 20 and 30° C.

The method can be carried out both under atmospheric pressure and. underelevated pressure of up to 2 bar, but it is preferably carried out underatmospheric pressure.

The invention is now additionally illustrated in detail by the followingexamples.

EXEMPLARY EMBODIMENTS Example 1 Preparation of the Lysine Oxidase fromPichia pastoris

1.a) Preparation of the Yeast Culture

-   -   3 g/l Difco malt extract,    -   5 g/l Difco peptone,    -   3 g/l Difco yeast extract and    -   20 g/l Difco agar        were cultivated on YM agar plates. For this purpose, the plates        were incubated at 28° C. for 48 hours and can then be kept at 4        to 5° C. for at least 4 weeks.        1.b) Preparation of the Preculture and of the Fermentation        Medium

-   b1) 10 g/l glucose are autoclaved at 121° C. for 30 minutes.

-   b2) 0.2 g/l MgSO₄.7H₂O,    -   3.0 g/l KH₂PO₄,    -   0.2 ml/l of a Vishniac & Santer salt solution containing 50 g/l        Titriplex III, 22 g/l ZnSO₄.7H₂O, 5.54 g/l CaCl₂, 5.06 g/l        MnCl₂.4H₂O, 4.99 g/l FeSO₄.7H₂O, ammonium heptamolybdate.4H₂O,        1.57 g/l CuSO₄.5H₂O, 1.61 g/l CoCl₂.6H₂O and adjusted with KOH        to pH 6.0,    -   were autoclaved at 121° C. for 30 minutes.

-   b3) 10 ml/l of a Lodder vitamin solution containing 20 μg/l biotin,    20 mg/l calcium pantothenate, 2 μg/l folic acid, 10 mg/l inositol,    200 μg/l p-aminobenzoic acid, 400 μg/l nicotinic acid, 400 μg/l    pyridoxine hydrochloride, 200 μg/l riboflavin and 400 μg/l thiamine    hydrochloride

were sterilized by filtration.

Medium ingredients b1) to b3) were combined after the sterilization.

500 ml of this fermentation medium were transferred into a 1 lErlenmeyer flask. From 3 to 4 agar plates containing the Pichia pastorisyeast culture (see l1.a)) the yeast cells were removed with aninoculation needle and transferred into said Erlenmeyer flask. Theincubation was carried out at 28° C. and 200 revolutions per minute for24 hours.

1.c) Preparation of the Main Culture

The preculture prepared in 1.b) was transferred into a 10 l fermenterand diluted with water. The pH was adjusted to about 7.0 by adding 50%by volume aqueous n-butylamine solution. The fermentation was carriedout at a temperature of 28° C. and with aeration at 5 l/min at 400revolutions per minute for about 28 to 30 hours. The time to stop thefermentation was found by determining the absorption at 600 nm (OD 600;OD=optical density). The OD 600 of the fermenter samples was measuredwith air as reference and was intended to be 0.9 to 1.0.

For workup, the contents of the fermenter were centrifuged at 45° C. and5 000 revolutions per minute (about 95 000 g; g=acceleration due to thegravity) for 20 minutes. The supernatant solution was removed, and theresidue was washed with 250 ml of 50 millimolar potassiumphosphate/potassium diphosphate buffer solution and centrifuged again.The supernatant solution was again removed, and the biomass remaining inthe residue was stored in a deepfreeze at −15° C.

1.d) Cell Disruption and Homogenization

18 ml of the Pichia pastoris biomass which had been stored at −20° C.and thawed were diluted with a buffer solution containing 20 mmol/lsodium phosphate and 1 mmol/l ethanolamine (pH 7.0) to 50 ml. 50 ofglass beads with a diameter of 0.5 mm were added to this solution, andthe mixture was homogenized at 5 000 revolutions per minute and 0° C.for 30 minutes. The homogenate was filtered through gauze. The filtratewas centrifuged at 8 000 revolutions per minute and 4° C. for 10minutes. The supernatant was removed.

Example 2 Purification of the Lysine Oxidase from Pichia pastoris

2.a) Ion Exchange Chromatography

The removed supernatant was adjusted to pH 7.0 with NaOH and loaded ontoa Q-Sepharose fast flow column from Pharmacia with a diameter of 5 cm, aheight of 13 cm and a volume of 250 ml. After loading, the column waswashed with 600 ml of solution A. Solution A contains sodium phosphatein a concentration of 20 mmol/l and ethanolamine in a concentration of 1mmol/l. Solution B contains sodium phosphate in a concentration of 20mmol/l, ethanolamine in a concentration of 1 mmol/l and NaCl in aconcentration of 1 mol/l. A linear gradient of 1 l of solution A and 1 lof solution B was used for elution. The active fractions were collected.

2.b) Molecular Sieve Chromatography

The active fractions were filtered through a 10 Da Omega filter(ultrafiltration) and separated on a preparative Superdex column fromPharmacia with a diameter of 2.6 cm, a length of 60 cm and a volume of320 ml. The mobile phase used was a solution containing 20 mmol/l sodiumphosphate/sodium diphosphate buffer, 150 mmol/l NaCl and 1 mmol/lethanolamine with a pH of 7.5. The mobile phase was passed through thecolumn at a rate of 3 ml/min. The active fractions were determined bythe method described under 2.d) and combined.

2.c) Ion Exchange Chromatogaphy

The combined active fractions obtained under 2.b) were further purifiedby chromatography on a Mono-Q HR5/5 column from Pharmacia. A mixture ofsolution A and solution B was used as mobile phase. This was passedthrough a column at a rate of 1 mil/min, and 100 fractions each of 1 mlwere collected. The active fractions were likewise determined by themethod described under 2.d) and combined. The lysine oxidase eluted asactive main protein. The protein had a molecular weight of about 121 000Da in an SDS gel under reducing conditions, for example through additionof β-mercaptoethanol or dithiothreitol.

2.d) Determination of the Lysine Oxidase Activity

The active fractions, that is to say the lysine oxidase activity weredetermined by utilizing the conversion of benzylamine into benzaldehydein the presence of lysine oxidase. Benzaldehyde is UV-active and can bedetected at 250 nm. An aliquot of the fractions collected under 2.a) to2.c) was incubated in each case with 3 mmol of benzylamine in aphosphate/dihydrogen phosphate buffer for 1 to 5 hours. The solutionobtained in this way was analyzed in an HPLC system using a Li-Chrosorb5RP C18 chromatography column from Merck. Water containing 0.1% byvolume of trifluoroacetic acid was used as mobile phase A, andacetonitrile containing 0.1% by volume of trifluoroacetic acid was usedas mobile phase B.

TABLE 1 HPLC program Mobile Flow Time phase rate [min] [% B] [ml/min] 040 1 6 70 1 6.1 100 1 6.5 100 1.5 9 40 1.5The amount of benzaldehyde produced was determined from a calibrationplot (1-100 μmol).2.e) Determination of the Amino Acid Sequence

In the Edman degradation of the lysine oxidase from Pichia pastoris, anamino-terminal sequence with the sequence SEQ ID NO 1 (see sequencelisting) was obtained. Partial sequences SEQ ID NO 2 to 12 (see sequencelisting) were obtained by proteolytic degradation of the lysine oxidasefrom Pichia pastoris with trypsin.

Example 3 Determination of the Enzymatic Activity in the Presence andAbsence of Reducing Agent

3.a) Assays with Various Substrates

The substrates employed were L-lysine monohydrochloride (for biochemicalpurposes, 99% from Merck), ethanolamine from BASF AG, glycine ethylester, 1,8-diaminooctane from Aldrich, n-butylamine from BASF AG,1,4-diaminobutane from BASF AG, ethylenediamine from BASF AG, sperminefrom Aldrich, 3-aminomethylpyridine from Aldrich, benzylamine fromAldrich, L-(+)-ornithine hydrochloride (99% from Aldrich) and1,6-diaminohexane from BASF AG.

Measurement series in each case with a concentration of 1 200, 600, 300,150, 75, 37.5 and 18.75 μmol of (di)amine per 1 l of water were carriedout in a microtiter plate. 2 measurement series were carried out in eachcase; one with enzyme and one without enzyme as reference.

100 μl of each of the diluted solutions were taken. To this solutionwere added 25 μl of lysine oxidase and 40 μl of 35 millimolar phenolsolution in 200 millimolar potassium phosphate/potassium diphosphatesolution with a pH of 7.5, 40 μl of a 2 millimolar aqueous4-aminoantipyrine solution and 40 μl of a solution of 0.35 mg ofperoxidase (horseradish from Sigma) in 1 ml of water.

In the reference measurement series, the 25 μl of lysine oxidase werereplaced by 25 μl of water. A violet dye was formed by the ammoniaproduced on conversion of the substrates and its absorption in the UV at500 nm was measured (T. Uwajima, O. Terada, Methods in Enzymology 1982,89, 243 ff.).

The relative conversion rates of the substrates compared with L-lysineare to be found in table 1.

TABLE 1 Relative conversion Substrate to be rate compared converted withL-lysine in % L-Lysine 100 α-N-Acetyllysine 120 D-Lysine 100D/L-5-Hydroxylysine 60 L-Ornithine 90 D-Ornithine 90 D/L-Ornithine 39difluoromethyl ester 1,6-Diaminohexane 180 Ethylenediamine 841,4-Diaminobutane 133 1,8-Diaminooctane 197 Spermine 185 n-Butylamine 63Ethanolamine 50 Glycine ethyl ester 93 3-Aminomethylpyridine 128Benzylamine 108

It emerged that both (un)substituted aliphatic amines and diamines, andamino acids and their derivatives can be converted in the presence oflysine oxidase. Remarkably, the conversion rate of some substrates isincreased by comparison with L-lysine. The use of derivatives of lysineand ornithine is of particular interest in relation to the preparationof piperidine-2-carboxylic acid and proline derivatives.

3.b) Assay with Various Concentrations of Reducing Agent

In order to test the tolerance of lysine oxidases to reducing agents, byway of example the measurement series with L-lysine using the lysineoxidase from Pichia pastoris was repeated in the presence of 0.001% byweight, 0.01% by weight, 0.1% by weight, 0.5% by weight and 1% by weightof NaBH₄ based on the total amount of substrate solution. The resultsare to be found in table 2.

TABLE 2 pH Lysine oxidase from during Conversion Pichia pastorisActivity in U/l incubation in % Without NaBH₄ 277 ~7 100.0 +0.001 wt. %NaBH₄ 271 ~7 97.8 +0.01 wt. % NaBH₄ 276 ~7 99.6 +0.1 wt. % NaBH₄ 276 ~799.6 +0.5 wt. % NaBH₄ 203 ~8.5 73.3 +1 wt. % NaBH₄ 167 ~9–10 60.3

It emerged that the activity of the lysine oxidase remained virtuallyunchanged up to a concentration of 0.1% by weight NaBH₄. Atconcentrations of ≧0.5% by weight NaBH₄, the activity decreased becauseof the increase in the pH. Relatively good conversions were stillachieved up to an NaBH₄ concentration of 1% by weight. Thus 1% by weightNaBH₄, based on the total amount of the substrate solution, was employedin each of the subsequent tests.

3.c) Workup of the Samples

50 ml of a 1.2 millimolar L-lysine solution converted in the presence oflysine oxidase was evaporated to dryness, taken up in five times theamount of ethanol, mixed with 1 drop of concentrated sulfuric acid andleft to stand at room temperature overnight. After removal of theethanol by distillation in vacuo, the residue was mixed with an excessof triethylamine and benzyl chloroformate and left to react at roomtemperature for 3 h. After addition of water, the N-benzyloxycarbonylderivatives wee extracted with dichloromethane. The organic phase wascharacterized by mass spectrometry and NMR. The resulting spectracorresponded to those of authentic samples of piperidine-2-carboxylicacid.

The other substrate solutions were worked up analogously.

1. A method for reacting amines of the general formula (I)

with A- meaning

to give compounds of the general formula (II) or (III)

with B- meaning HO—CH₂—R³— or R⁴— comprising the following steps: a)oxidation of at least one

 group of an amine of the general formula (I) to a carbonyl group in thepresence of a lysine oxidase as catalyst; b) where appropriate reactionof the second NH₂ group of an amine of the general formula (I) with

 with the carbonyl group resulting from a) to give an enamine or imineby cyclization; c) reduction of the carbonyl group(s) resulting from a),or of the anamine or imine resulting where appropriate from b), with areducing agent respectively to hydroxymethyl groups or to a cyclicsecondary amine of the general formula (II), where R¹ is NR⁵ or a linearbivalent C₂–C₆ hydrocarbon radical which is optionally substituted byCO₂H, C₂R⁶, OH, SH and/or N(R⁵)₂ and/or contains nonadjacent O, S and/orN atoms, where R⁵ is H or linear C₁–C₆-alkyl and R⁶ is linearC₁–C₆-alkyl or branched C₃–C₆-alkyl or C₆–C₁₀-aryl, and R² is H, CO₂H,C₂R⁶ or CN, where the method is carried out as a one-pot method, lysineoxidise and reducing agent are present together, and in the definitionof A in the general formula (I) R³ is a bivalent C₀–C₁ or C₅–C₁₈hydrocarbon radical which is optionally substituted by linearC₁–C₆-alkyl or branched C₃–C₆-alkyl, C₆–C₁₀-aryl, C₄–C₁₀-heteroaryland/or CO₂R⁶ and/or contains nonadjacent O, S and/or N atoms, and R⁴ isa linear C₁–C₂₀ hydrocarbon radical which is optionally substituted byfunctional groups CO₂H and/or CO₂R⁶ and/or contains nonadjacent O, Sand/or N atoms.
 2. A method for preparing cyclic secondary amines of thegeneral formula (II) from diamines of the general formula (Ia)

where R¹ is NR⁵ or a linear bivalent hydrocarbon radical with 2 to 6 Catoms, which is optionally substituted by functional groups CO₂H,C₂R^(6,) OH, SH and/or N(R⁵)_(2,) and/or contains nonadjacentheteroatoms O, S and/or N, p1 where R⁵ is H or linear C₁–C₆-alkyl and R⁶is linear C₁–C₆-alkyl or branched C₃–C₆-alkyl or C₆–C₁₀-aryl, and R² isH, CO₂H, C₂R⁶ or CN, comprising the following steps: oxidation of one ofthe two

 groups of a dimaine of the general formula (Ia) to a carbonyl group inthe presence of a lysine oxidase as catalyst; b) reaction of the secondNH₂ group of the diamine of the general formula (Ia) with the carbonylgroup resulting from step a) to give an enamine or imine by cyclization;c) reduction of the enamine or imine resulting from b) with a reducingagent to give a cyclic secondary amine of the general formula (II);wherein the method is carried out as one-pot method, and lysine oxidaseand reducing agent are present together.
 3. A method as claimed in claim2, wherein the diamine of the general formula (Ia) is selected from thegroup consisting of lysine, omithine and 1,6-diaminohexane.
 4. A methodfor preparing diols of the general formula (IIIa) from diamines of thegeneral formula (Tb)

comprising the following steps: a) oxidation of the two aminomethylgroups of a diamine of the general formula Ib) to formyl groups in thepresence of a lysine oxidizase as catalyst; b) reduction of the formylgroups resulting from a) with a reducing agent to hydroxymethyl groupsto form a diol of the general formula (IIIa); wherein the method iscarried out as one-pot method, lysine oxidize and reducing agent arepresent together, and R³ is a bivalent C₀–C₁ or C₅–C₁₈ hydrocarbonradical which is optionally substituted by linear C₁–C₆-alkyl orbranched C₃–C₆-alkyl, C₆–C₁₀-aryl, C₄–C₁₀-heteroaryl and/or CO₂R^(6,)and/or contains nonadjacent O, S and/or N atoms, wherein R⁶ is linearC₁–C₆-alkyl or branched C₃–C₆-alkyl or C₆–C₁₀-aryl.
 5. A method forpreparing primary alcohols R⁴—CH₂—OH from amines R⁴—CH₂—NH₂ comprisingthe following steps: a) oxidation of the aminomethyl group of an amineR⁴—CH₂—NH2 to a formyl group in the presence of a lysine oxidase ascatalyst; b) reduction of the formyl group resulting from a) with areducing agent to a hydroxylmethyl group to form a primary alcoholR⁴—CH₂—OH; wherein the method is carried out as one-pot method, lysineoxidase and reducing agent are present together, and R⁴ is a linearC₁–C₂₀ hydrocarbon radical which is optionally substituted by functionalgroups CO₂H and/or CO₂R⁶ and/or contains nonadjacent O, S and/or Natoms, wherein R⁶ is linear C₁–C₆-alkyl or branched C₃–C₆-alkyl orC₆–C₁₀-aryl.
 6. A method as claimed in claim 1, wherein a lysine oxidasefrom a yeast is employed.
 7. A method as claimed in claim 2, wherein alysine oxidase from a yeast is employed.
 8. A method as claimed in claim3, wherein a lysine oxidase from a yeast is employed.
 9. A method asclaimed in claim 4, wherein a lysine oxidase from a yeast is employed.10. A method as claimed in claim 5, wherein a lysine oxidase from ayeast is employed.
 11. The method of any of claims 6, 7, 8, 9, or 10,wherein said yeast is Pichia pastoris.